Agent for inducing callus and method for inducing callus

ABSTRACT

This invention relates to an agent for inducing a callus comprising a compound represented by Formula (I) or a hydrolysis product of an amide bond thereof: 
     
       
         
         
             
             
         
       
     
     wherein Ar 1  represents phenyl substituted with substituent or substituents selected from alkoxy and methylenedioxy; Ar 2  represents phenyl substituted with halogen; R 1  and R 2  each represent hydrogen, alkyl, cyano, or carboxyl; R 1  and R 2  may together form oxo; R 3  to R 10  each represent hydrogen or methyl; and R 3  and R 4 , R 5  and R 6 , R 7  and R 8 , and/or R 9  and R 10  may together form oxo; a method for inducing a callus and a method for plant transformation using such agent for inducing a callus.

TECHNICAL FIELD

The present invention relates to an agent for inducing a callus and amethod for inducing a callus.

BACKGROUND ART

Plants have totipotency, such that they are able to form calluses fromsomatic cells that have been highly differentiated. If calluses arecultured under constant conditions, also, plant bodies can be reproducedthrough differentiation of adventitious embryos, adventitious buds, andadventitious roots. Callus culture is advantageous in the followingrespect; for example, 1) it has reproductive integrity; 2) it has theability to differentiate a callus cell mass into various tissues andindividuals; 3) a large quantity of homogeneous growth cells that aredifficult to obtain in plants can be obtained; 4) a callus is suitableas an experimental material because of the absence of seasonal changesin the quality or quantity of cells; 5) the influence of a substancethat has been added to the medium on plants can be directly observed;and 6) some plant species are easily induced to undergo geneticvariation as a result of callus formation, and such plants can be usedfor breeding. Thus, callus culture has been extensively used for theproduction of useful materials, the development of new varieties, thegene introduction into plants, the reproduction of transformants, theproduction of artificial seeds, and other purposes.

In general, a callus is produced by a method in which a piece of planttissue is cultured in a medium containing phytohormones (i.e., auxin andcytokinine) (Skoog, F., and Miller, C. O., 1957, Chemical regulation ofgrowth and organ formation in plant tissue cultured in vitro, Symp. Soc.Exp. Biol., 54, 118-130). This method was established over 30 years ago,and this technique is indispensable for the production of transgeniccrops at present. However, types of auxin and cytokinine to be used andthe amount of auxin relative to that of cytokinine vary depending onplant species. Accordingly, it has been difficult to find adequateconditions for callus formation.

As synthetic auxins, 2,4-dichlorophenoxyacetic acid and2,4,5-trichlorophenoxy acetic acid are known, and, concerning an analogthereof; i.e., 4-chlorophenoxyacetic acid, it was reported that thenumber of days required for leaf tissue culture of African violet, fromimplanting of explants to callus formation, and formation ofadventitious shoots and adventitious roots, was reduced as a result oftreatment performed at a particular concentration (Miyoshi Hakozaki etal., Effects of 4-Chlorophenoxyacetic Acid on the Callus andAdventitious Organogenesis from Cultured Leaf Explant African Violet inVitro, Memoirs of the Institute of Science and Technology, MeijiUniversity, 40, 1-7, 2001). However, callus formation efficiency was nothigh, and plant species capable of callus formation were limited.

As a 1,4-disubstituted piperazine derivative, JP H6-67668 (B) (1994)describes that N-methyl-N′-phenylacetylpiperazine,N-benzyl-N′-benzoylpiperazine, and the like are useful as decolorantsagainst the color development system of bicolor thermal recordingmaterials.

JP 2002-503239 (A) describes that a piperazine derivative havingsubstituents at positions other than 1- and 4-positions of thepiperazine ring is useful as an anti-inflammatory agent.

Fipexide, which is a type of a 1,4-disubstituted piperazine derivative(e.g., 1-[(p-chlorophenoxy)acetyl]-4-piperonylpiperazine or1-(4-chlorophenoxyacetyl)-4-(1,3-benzodioxole-5-ylmethyl)piperazine), isknown as an antidepressant.

Further, 1-piperonylpiperazine (1-(3 ,4-methylenedioxybenzyl)piperazine)and many 1-substituted piperazine derivatives as analogs thereof arecommercially available as raw materials for chemical production.

However, there has been no report demonstrating that 1,4-disubstitutedpiperazine derivatives and 1-substituted piperazine derivatives arecapable of callus induction.

SUMMARY OF THE INVENTION Objects to Be Attained by the Invention

It is an object of the present invention to provide an agent forinducing a callus, which has a basic structure different from that of aconventional agent for inducing a callus, and a method for inducing acallus using such agent for inducing a callus.

Means for Attaining the Objects

The present invention is summarized as follows.

(1) An agent for inducing a callus comprising a compound represented byFormula (I) or a salt thereof:

wherein Ar¹ represents a phenyl group substituted with at least onesubstituent selected from among a C₁₋₆-alkoxy group and a substituted orunsubstituted methylenedioxy group;

Ar² represents a phenyl group substituted with 1 to 3 halogen atoms;

R¹ and R² each represent a hydrogen atom, a substituted or unsubstitutedC₁₋₃-alkyl group, a cyano group, or a carboxyl group;

R¹ and R² may together form an oxo group;

R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ each represent a hydrogen atom or amethyl group; and

R³ and R⁴, R⁵ and R⁶, R⁷ and R⁸, and/or R⁹ and R¹⁰ may together form anoxo group.

(2) The agent for inducing a callus according to item (1) above, whereinthe compound represented by Formula (I) or a salt thereof is fipexide ora salt thereof.(3) An agent for inducing a callus comprising a compound represented byFormula (I-1) or a salt thereof:

wherein Ar¹, R¹, R², R³, R⁴, R³, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are as definedabove with respect to Formula (I) in item (1); and R¹ represents ahydrogen atom, a substituted or unsubstituted C₁₋₇ hydrocarbon group, oran amidino group, and a compound represented by Formula (I-2) or a saltthereof:

HO—CO—CH₂—O—Ar²  (1-2)

wherein Ar² is as defined above with respect to Formula (I) in item (1).(4) The agent for inducing a callus according to item (3), wherein thecompound represented by Formula (I-1) or a salt thereof is1-piperonylpiperazine or a salt thereof and the compound represented byFormula (I-2) or a salt thereof is 4-chlorophenoxyacetic acid or a saltthereof.(5) A method for inducing a callus comprising bringing a plant, a plantcell, a piece of plant tissue, or a plant seed into contact with theagent for inducing a callus according to any of (1) to (4) and inducingcallus formation.(6) A method for producing a callus comprising bringing a plant, a plantcell, a piece of plant tissue, or a plant seed into contact with theagent for inducing a callus according to any of items (1) to (4),inducing callus formation, and growing the callus.(7) A callus produced by the method according to item (6).(8) A method for plant transformation involving the use of a mediumcontaining the agent for inducing a callus according to any of items (1)to (4) as a callus induction medium in the method for planttransformation by the Agrobacterium method.

EFFECTS OF THE INVENTION

The agent for inducing a callus according to the present inventioncomprises, as an active ingredient, a 1,4-disubstituted piperazinederivative or a 1-substituted piperazine derivative having a basicstructure different from a conventional agent for inducing a callus.Thus, such agent can be applied to a wide variety of plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of the callus induction activity test offipexide (FPX) using Arabidopsis.

FIG. 2 shows the redifferentiation conditions as a result of treatmentwith fipexide (A) and without treatment (B).

FIG. 3 shows the results of a comparison of callus induction efficiencyattained with the aid of fipexide (FPX) and that attained via aconventional technique (with the aid of 2,4-dichlorophenoxyacetic acid(2,4-D)/kinetin).

FIG. 4 shows the results of the callus induction activity tests offipexide (FPX), 4-chlorophenoxyacetic acid (CPA) alone,1-piperonylpiperazine (PPZ) alone, and 4-chlorophenoxyacetic acid (CPA)in combination with 1-piperonylpiperazine (PPZ).

FIG. 5 shows the results of the callus induction activity test offipexide (FPX) using the wild-type rice (Nipponbare).

FIG. 6 shows the results of the callus induction activity test offipexide (FPX) on soybean seed (Tsurunoko), tomato seed (Micro-Tom), andcucumber seed (Natsu Suzumi).

FIG. 7 shows the results of the callus induction activity test offipexide (FPX) using wild-type Populus.

FIG. 8 shows the results of plant transformation performed by theAgrobacterium method that employs callus induction caused by fipexide.

EMBODIMENTS FOR IMPLEMENTING THE INVENTION

Hereafter, the present invention is described in detail.

The present invention relates to an agent for inducing a callus and amethod for inducing a callus with the use of a compound comprising aparticular type of substituted piperazine skeleton in which a nitrogenatom is substituted with Ar¹—C(R¹)(R²)— and a phenoxyacetyl group(—CO—CH₂—O—Ar²) in which a benzene ring is substituted with 1 to 3halogen atoms in a molecule or with the use of two types of compoundscomprising the substituted piperazine skeleton and the substitutedphenoxyacetyl group in separate molecules in combination.

The correlation between the compound comprising the substitutedpiperazine skeleton and callus induction has not yet been reported.

Examples of halogen atoms as substituents of the substituted phenylgroup represented by Ar² in Formulae (I) and (I-2) include fluorine,chlorine, and iodine atoms.

Examples of compounds comprising a phenoxyacetyl group (—CO—CH₂—O—Ar²)with a benzene ring being substituted with 1 to 3 halogen atoms include2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, and4-chlorophenoxyacetic acid, and these compounds are known as syntheticauxins.

Examples of the C₁₋₆-alkoxy group as a substituent of the substitutedphenyl group represented by Ar¹ in Formulae (I) and (I-1) include amethoxy group, an ethoxy group, a propoxy group, and an isopropoxygroup, and examples of the substituted methylenedioxy group include adifluoromethylenedioxy group and a dichloromethylenedioxy group.

Examples of the substituted phenyl group represented by Ar¹ include a3,4-methylenedioxyphenyl group (a 1,3-benzodioxol-5-yl group), a2,3-methylenedioxyphenyl group (a 1,3-benzodioxol-4-yl group), a3,4-(difluoromethylenedioxy)phenyl group (a2,2-difluoro-1,3-benzodioxol-5-yl group), a2,3-(difluoromethylenedioxy)phenyl group (a2,2-difluoro-1,3-benzodioxol-4-yl group), a3,4-methylenedioxy-5-methoxyphenyl group (a7-methoxy-1,3-benzodioxol-5-yl group), a 3,4-dimethoxyphenyl group, anda 3,4,5-trimethoxyphenyl group, with the 3,4-methylenedioxyphenyl group(the 1,3-benzodioxol-5-yl group) being preferable.

Examples of the substituted phenyl group represented by Ar² in Formulae(I) and (I-2) include a 4-chlorophenyl group, a 2,4-dichlorophenylgroup, and a 2,4,5-trichlorophenyl group, with the 4-chlorophenyl groupbeing preferable.

Examples of the C₁₋₃-alkyl group represented by R¹ or R² in Formulae (I)and (I-1) include a methyl group, an ethyl group, a propyl group, and anisopropyl group, and such C₁₋₃ alkyl group may be substituted with oneor more substituents selected from among, for example, an amino group, ahydroxyl group, a carboxyl group, a cyano group, a halogen atom (e.g., afluorine, chlorine, or iodine atom), and a nitro group. R¹ and R² maytogether form an oxo group. R¹ and R² are preferably hydrogen atoms.

In Formulae (I) and (I-1), R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ arepreferably hydrogen atoms.

Examples of the C₁₋₇ hydrocarbon group represented by R¹¹ in Formula(I-1) include: C₁₋₅-alkyl groups such as a methyl group, an ethyl group,a propyl group, an isopropyl group, a butyl group, an isobutyl group, asec-butyl group, a tert-butyl group, a pentyl group, and an isopentylgroup; C₂₋₅-alkenyl groups such as an allyl group (a 2-propen-1-ylgroup) and a 2-methyl-2-propen-1-yl group; C₂₋₅-alkynyl groups such as apropargyl group (a 2-propyn-1-yl group); and a benzyl group. Such C₁₋₇hydrocarbon group may be substituted with one or more substituentsselected from among, for example, an amino group, a hydroxyl group, acyano group, a halogen atom (e.g., a fluorine, chlorine, or iodineatom), and a methoxy group.

Among the compounds represented by Formula (I), fipexide in which asubstituted phenyl group represented by Ar¹ is a3,4-methylenedioxyphenyl group (a 1,3-benzodioxol-5-yl group), asubstituted phenyl group represented by Ar² is a 4-chlorophenyl group,and R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are each a hydrogen atomis a commercially available compound known as an antidepressantcomprising a small number of substituents. This compound is preferablefrom the viewpoint of availability.

Among the compounds represented by Formula (I-1), 1-piperonylpiperazinein which a substituted phenyl group represented by Ar¹ is a3,4-methylenedioxyphenyl group (a 1,3-benzodioxol-5-yl group) and R¹,R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹ are each a hydrogen atom isa commercially available compound comprising a small number ofsubstituents. This compound is preferable from the viewpoint ofavailability.

Examples of salts of the compounds represented by Formula (I) or (I-1)include salts with inorganic acids, such as hydrochloric acid, sulfuricacid, phosphoric acid, hydrobromic acid, hydroiodic acid, nitric acid,pyrosulfuric acid, and metaphosphoric acid, and salts with organicacids, such as citric acid, benzoic acid, acetic acid, propionic acid,fumaric acid, maleic acid, tartaric acid, succinic acid, sulfonic acid(e.g., methanesulfonic acid, p-toluenesulfonic acid, ornaphthalenesulfonic acid), and amino acid (e.g., glutamic acid).

Examples of salts of the compounds represented by Formula (I-2) includealkaline metal salts, such as sodium salt and potassium salt, lysinesalt, and arginine salt.

The compounds represented by Formula (I) can be produced in accordancewith a conventional technique, such as the method described in JP2002-503239 A, in the manner described below:

wherein Ar¹, Ar², R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are asdefined above; and X¹ and X² each independently represent a halogen atom(a chlorine, bromine, or iodine atom).

A compound represented by Formula (I-1a) is equivalent to a compoundrepresented by Formula (I-1), wherein R¹¹ represents a hydrogen atom.

Products that can be obtained in the manner described above may bepurified in accordance with a conventional technique, such as columnchromatography using silica gel as a carrier, or recrystallization usingmethanol, ethanol, chloroform, dimethylsulfoxide, or water. Examples ofeluting solvents used for column chromatography include methanol,ethanol, chloroform, acetone, hexane, dichloromethane, ethyl acetate,and a solvent mixture of any thereof.

Many of the compounds represented by Formulae (I), (I-1), and (I-2) arecommercially available, and such commercially available compounds can beused in the present invention.

Examples of plants to which the present invention is applicable include:dicotyledonous plants, such as Arabidopsis plants, trees (e.g., Populusand Eucalyptus), rapeseed, tomato, tobacco, soybean, carrot, melon,apple, Manihot utilissima, Spirodela polyrhiza, and Striga; andmonocotyledonous plants, such as Gramineae plants (e.g., rice, wheat,barley, maize, and Brachypodium distachyon) and Liliaceae plants (e.g.,onion).

Plants, plant cells, pieces of plant tissue, or plant seeds to beimplanted into media are not particularly limited, provided that theyare capable of callus induction. Examples of plant tissues include shootapex, stalk, leaf, shoot, germ cell, and root. Plants and the like to beimplanted are preferably disinfected and sterilized with the use of, forexample, an aqueous solution of sodium hypochlorite. Plants and the likegrown by aseptic seeding are not necessarily sterilized.

The agent for inducing a callus according to the present invention cancontain known additives for preparation, in addition to the activeingredients described above. Examples of known additives for preparationinclude an excipient, an emulsifier, and a moistening agent. Theconfiguration of the agent for inducing a callus according to thepresent invention is not particularly limited, provided that such agentcan be used in the art. Examples of configurations include an emulsion,a liquid, an oil solution, an aqueous solution, water dispersiblepowder, a flowable agent, a dusting powder, a microgranule, a granule,an aerosol, and a paste.

In the method for inducing a callus and the method for producing acallus according to the present invention, plants and the like can bebrought into contact with the agent for inducing a callus according tothe present invention without particular limitation. In accordance witha plant type, a target organ, a dosage form of the agent for inducing acallus, and other factors, an adequate means can be selected from amongsoaking, coating, spraying, and addition to the medium. To this end,plants and the like are preferably cultured in a medium containing theagent for inducing a callus according to the present invention.

Any callus induction medium may be used without particular limitation,provided that it contains (i) the compound represented by Formula (I) ora salt thereof or (ii) the compound represented by Formula (I-1) or asalt thereof and the compound represented by Formula (I-2) or a saltthereof and it is capable of callus induction.

While the concentration of the compound represented by Formula (I) or asalt thereof in the medium is not particularly limited, it is generally5 to 200 μM and preferably 15 to 60 μM.

When the compound represented by Formula (I-1) or a salt thereof is usedin combination with the compound represented by Formula (I-2) or a saltthereof, the concentration of the compound represented by Formula (I-1)or a salt thereof in the medium is not particularly limited, and it isgenerally 0.01 to 100 μM, and preferably 0.1 to 60 μM. While theconcentration of the compound represented by Formula (I-2) or a saltthereof in the medium is not particularly limited, it is generally 0.01to 100 μM, and preferably 0.1 to 60 μM.

Components other than the compound described above may be, for example,a saccharide, a gelling agent, or an inorganic salt, which is generallyused for callus induction. Phytohormone may be added to the medium,provided that the effects of the present invention are not adverselyaffected.

Culture is preferably conducted under aseptic conditions. Culture ispreferably carried out at 20° C. to 25° C., and light conditions arepreferably set between continuous light conditions and continuous darkconditions. In general, callus induction is observed 2 to 4 weeks afterthe initiation of culture.

In order to grow the callus that was induced in the manner describedabove, for example, the medium may be exchanged with a fresh callusinduction medium (i.e., agarose solid MS medium or liquid MS mediumcontaining 0.9% sucrose) every month.

The grown callus may be redifferentiated by, for example, transferringthe callus to a MS medium (0.9% agarose and 1.5% sucrose) containingauxin (indoleacetic acid) at 0.15 mg/l and cytokinine(N⁶-2-isopentenyladenine) at 0.5 mg/l and developing a shoot (anadventitious bud) about 2 to 4 weeks thereafter.

The callus obtained according to the present invention can be used forproduction of a transgenic crop. For example, a callus may be preparedfrom a cell infected with Agrobacterium, or a callus may be infectedwith Agrobacterium comprising a plasmid to be introduced into a calluscell. Thereafter, a cell comprising a plasmid inserted into thechromosome may be separated and then redifferentiated to reproduce aplant. Thus, a transgenic crop that is capable of stable gene transfercan be obtained.

This description includes part or all of the content as disclosed in thedescription and/or drawings of Japanese Patent Application No.2014-117832, which is a priority document of the present application.

EXAMPLES

Hereafter, the present invention is described in greater detail withreference to the examples, although the technical scope of the presentinvention is not limited to these examples.

[Example 1] Fipexide Activity Test using Arabidopsis

(1) Morphological Observation of Arabidopsis (Columbia) by Treatmentwith Fipexide

Wild-type seeds of Arabidopsis (Columbia) were sowed in a 1/2 MS mediumsupplemented with fipexide at 0, 15, 30, or 45 μM, and morphologicalobservation was conducted.

As a result of treatment with fipexide, callus formation was mainlyobserved in the root at low concentration of 15 μM and in the shoot apexat high concentration of 45 μM (FIG. 1). Since callus formation issuppressed in the root at high concentration, it was considered that theroot was more sensitive to fipexide than the shoot apex and celldivision was inhibited in the root.

(2) Callus Induction and Redifferentiation by Treatment with Fipexide

The root organ of Arabidopsis (Columbia) was cut, treated in a ½ MSmedium supplemented with fipexide at 45 μM for 2 weeks to induce acallus, and then cultured in a redifferentiation medium(auxin/cytokinine). As a result, redifferentiation was observed (FIG.2A).

In contrast the root organ of Arabidopsis (Columbia) was cut and thencultured in the redifferentiation medium without callus induction. As aresult, redifferentiation was not observed (FIG. 2B).

(3) Comparison of Fipexide with Conventional Technique

Callus induction in Arabidopsis that was carried out at the optimizedauxin/cytokinine concentration (i.e., 2,4-dichlorophenoxyacetic acid at2.26 μM and kinetin at 0.465 μM) was compared with callus induction thatwas carried out at the fipexide concentration of 45 μM. As a result,callus induction efficiency of the fipexide was found to be higher thanthat attained at the optimized auxin/cytokinine concentration. FIG. 3shows the conditions of the hypocotyl that had been cultured underconstant lighting conditions for 87 days.

(4) Functional Analysis of Putative Fipexide Metabolites (i.e.,4-chlorophenoxyacetic Acid and 1-piperonylpiperazine)

Fipexide is a compound formed by an amide bond between4-chlorophenoxyacetic acid and 1-piperonylpiperazine, and fipexide isdeduced to be non-enzymatically hydrolyzed in a plant. Thus, treatmentwith 4-chlorophenoxyacetic acid or 1-piperonylpiperazine alone ortreatment with both thereof was attempted.

Arabidopsis (Columbia) was allowed to germinate under weak lightingconditions for 7 days, the hypocotyl cut therefrom was placed on a 1/2MS medium supplemented with 4-chlorophenoxyacetic acid and/or1-piperonylpiperazine at 0, 5, 15, 30, 45, or 60 μM, and morphologicalobservation thereof was conducted.

As a result, callus induction activity was observed by treatment with4-chlorophenoxyacetic acid as with the case of treatment with fipexide.This indicates that the 4-chlorophenoxyacetic acid structure plays a keyrole in callus formation.

When the plant was treated with 1-piperonylpiperazine alone, incontrast, the number and the length of lateral roots were increased, andthe leaf area was enlarged, although callus induction activity was notobserved.

When the hypocotyl of Arabidopsis was treated with both4-chlorophenoxyacetic acid and 1-piperonylpiperazine, in addition, thecallus growth rate was faster than the case in which the plant wastreated with 4-chlorophenoxyacetic acid alone. This indicates that cellelongation activity of 1-piperonylpiperazine additively accelerates thecallus induction caused by 4-chlorophenoxyacetic acid.

The results are shown in FIG. 4.

[Example 2] Fipexide Activity Test using Other Plants

As with the case of Arabidopsis germination conditions, plants werecultured in a fipexide medium with the use of sterilized seeds. As aresult, callus induction was observed in germinated rice seeds,germinated Eucalyptus seeds, soybean seed (Tsurunoko), tomato seed(Micro-Tom), and cucumber seed (Natsu Suzumi).

FIG. 5 shows the conditions of the wild-type rice (Nipponbare) 30 daysafter seed germination. Callus formation was observed at fipexideconcentration of 45 μM.

FIG. 6 shows the results of callus induction activity tests of fipexide(FPX) on soybean seed (Tsurunoko), tomato seed (Micro-Tom), and cucumberseed (Natsu Suzumi).

As with the case of Arabidopsis plants, stalks and roots of Populus werecultured in a fipexide medium and, as a result, callus induction wasobserved in the stalks and the roots of Populus.

The induced callus was cultured in a redifferentiation medium (i.e., aMS medium (0.9% agarose and 1.5% sucrose) containing auxin (indoleaceticacid) at 0.15 mg/l and cytokinine (N⁶-2-isopentenyladenine) at 0.5 mg/lor a ½ MS medium (0.9% Phytoagar (agarose for plants) and 0.2% sucrose)containing 3-indolebutyric acid (IBA) at 0.1 mg/l and6-benzylaminopurine (BAP) at 0.2 mg/l). As a result, redifferentiationwas observed.

The conditions of the aseptic stalk (which is not the hypocotyl) and theroot organ sections of wild-type Populus 30 days after cutting in thepresence of fipexide are shown at the center of FIG. 7, and theconditions thereof 30 days after the medium was exchanged with a freshredifferentiation medium are shown on the right of FIG. 7.

[Example 3] Transformation Utilizing Callus Induction caused by Fipexide

Under general callus induction conditions for Populus, callus inductionwas conducted with the use of fipexide instead of auxin/cytokinine, andwhether or not Agrobacterium-infected callus was transformed wasexamined.

The GUS gene was introduced into a pH35GS binary vector comprising aGateway cassette (Kubo M. et al., Genes & Dev., 19, 1855-1860, 2005)(manufactured by Inplanta Innovations Inc.; Production code: IN3-VEC17)using the Gateway system, and the resulting binary vector was used forthe Agrobacterium method, so as to confirm infection.

The transformation procedure is described below.

1. From young plants of aseptically cultured Populus (Populustremula×tremuloides T89) that had been grown in pots, stalk samples(length: 5 mm) were cut.2. The stalk samples were cultured in the presence of Agrobacterium (for3 days, in the dark, at 22° C.).3. After Agrobacterium was washed away, the stalk samples were placed onMS1 medium or modified MS1 medium (containing 30 μM fipexide instead of3-indolebutyric acid (IBA) and 6-benzylaminopurine (BAP)) and culturedat 25° C.4. (On the fourth week, it was confirmed that many calluses were formedin modified MS1 medium containing fipexide while callus was not formedin conventional MS1 medium.)5. Two months after the initiation of callus induction culture, 23calluses were fixed in 90% (w/w) acetone at −30° C. overnight, thesamples were washed twice with 50 mM PBS buffer (pH 7.0), and thesamples were subjected to incubation in GUS substrate solution at 37° C.for 15 minutes for staining.

As a result, GUS activity was observed in all samples. The results areshown in FIG. 8.

The compositions of the MS1 medium, the modified MS1 medium, and the GUSsubstrate solution are shown below.

[MS1 medium composition (in 1 liter)]4.4 g of Murashige & Skoog salt20 g of sucrose

0.2 mg of BAP 0.1 mg of IBA

0.01 mg of TDZ (thidiazuron)pH 5.6[Modified MS1 medium composition (in 1 liter)]4.4 g of Murashige & Skoog salt20 g of sucrose30 μM of fipexidepH 5.6[GUS substrate solution composition (in 1 liter)]

1 mM of X-Gluc 50 mM of PBS (pH7.0) 0.1% of Triton X-100

1 mM of potassium ferricyanide1 mM of potassium ferrocyanide

Thus, it was found that callus induction caused by fipexide wasapplicable to a plant transformation technique by the Agrobacteriummethod.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

1. An agent for inducing a callus comprising a compound represented byFormula (I) or a salt thereof:

wherein Ar¹ represents a phenyl group substituted with at least onesubstituent selected from among a C₁₋₆-alkoxy group and a substituted orunsubstituted methylenedioxy group; Ar² represents a phenyl groupsubstituted with 1 to 3 halogen atoms; R¹ and R² each represent ahydrogen atom, a substituted or unsubstituted C₁₋₃-alkyl group, a cyanogroup, or a carboxyl group; R¹ and R² may together form an oxo group;R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ each represent a hydrogen atom or amethyl group; and R³ and R⁴, R³ and R⁶, R⁷ and R⁸, and/or R⁹ and R¹⁰ maytogether form an oxo group.
 2. The agent for inducing a callus accordingto claim 1, wherein the compound represented by Formula (I) or a saltthereof is fipexide or a salt thereof.
 3. An agent for inducing a calluscomprising a compound represented by Formula (I-1) or a salt thereof:

wherein Ar¹, R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are as definedabove with respect to Formula (I) in claim 1; and R¹¹ represents ahydrogen atom, a substituted or unsubstituted C₁₋₇ hydrocarbon group, oran amidino group, and a compound represented by Formula (I-2) or a saltthereof:HO—CO—CH₂—O—Ar²  (I-2) wherein Ar² is as defined above with respect toFormula (I) in claim
 1. 4. The agent for inducing a callus according toclaim 3, wherein the compound represented by Formula (I-1) or a saltthereof is 1-piperonylpiperazine or a salt thereof and the compoundrepresented by Formula (I-2) or a salt thereof is 4-chlorophenoxyaceticacid or a salt thereof.
 5. A method for inducing a callus comprisingbringing a plant, a plant cell, a piece of plant tissue, or a plant seedinto contact with the agent for inducing a callus according to any oneof claims 1 to 4 and inducing callus formation.
 6. A method forproducing a callus comprising bringing a plant, a plant cell, a piece ofplant tissue, or a plant seed into contact with the agent for inducing acallus according to any one of claims 1 to 4, inducing callus formation,and growing the callus.
 7. A callus produced by the method according toclaim
 6. 8. A method for plant transformation involving the use of amedium containing the agent for inducing a callus according to any oneof claims 1 to 4 as a callus induction medium in the method for planttransformation by the Agrobacterium method.